DNA Damage Response After Treatment of Cycling and Quiescent Cord Blood Hematopoietic Stem Cells With Distinct Genotoxic Noxae.

Hematopoietic stem cells (HSC) from cord blood can be applied as an alternative to bone marrow in transplantation to treat hematological diseases. Umbilical cord blood (UCB) consists of cycling and non-cycling CD34+/CD45low cells needed for long-term and short-term engraftment. After sorting and subsequent in vitro culture, quiescent HSCs enter the cell cycle. This enables the analysis of HSCs in 2 different cell cycle stages and the comparison of their responses to different genotoxic noxae. To analyze different mechanisms of DNA damage induction in cells, 2 different genotoxins were compared: etoposide, a topoisomerase II inhibitor that targets mitosis in the S/G2-phase of the cell cycle and the alkylating nitrosamine N-Nitroso-N-methylurea (MNU), which leads to the formation of methyl DNA adducts resulting in DNA double breaks during DNA replication and persistent mutations. Cycling cells recovered after treatment even with higher concentrations of etoposide (1.5µM/ 5µM/10µM), while sorted cells treated with MNU (0.1mM/0.3mM/0.5mM/1mM/3Mm/ 5mM) recovered after treatment with the lower MNU concentrations whereas high MNU concentrations resulted in apoptosis activation. Quiescent cells were not affected by etoposide treatment showing no damage upon entry into the cell cycle. Treatment with MNU, similarly to the cycling cells, resulted in a dose-dependent cell death. In conclusion, we found that depending on the genotoxic trigger and the cycling status, CD34+cells have distinct responses to DNA damage. Cycling cells employ both DDR and apoptosis mechanisms to prevent damage accumulation. Quiescent cells predominantly undergo apoptosis upon damage, but their cell cycle status protects them from certain genotoxic insults.

Long-Term Stability of Cord Blood Units After 29 Years of Cryopreservation: Follow-Up Data From the José Carreras Cord Blood Bank.

The José Carreras Cord Blood Bank (CBB) located in Düsseldorf as of today stores 21 215 active cryopreserved cord blood units (CBUs) applicable as a source for hematopoietic stem cell (HSC) transplantation. Since the success of transplantation outcomes is mainly dependent on the cord blood quality, typical parameters are evaluated by a Stability Monitoring Program specified by the FACT Standards. The longest expiration time determined to date is 29 years for unseparated units, 25 years for manual and 18 years for automated volume-reduced units licensed by the Paul-Ehrlich Institute. According to the CBB stability program TNC count, TNC recovery, TNC viability, CD34+7AAD- viability, CD45+7AAD- viability and CFC count were determined for all 3 processing methods applied over time. As a measure of stability, unseparated units (processed 1993-1998) revealed a mean TNC viability of 88.91 ±â€…5.01% after 29 years of cryopreservation versus manual volume-reduced CBUs (processed 1998-2005) with a mean of 84.22 ±â€…10.02% after 25 years of cryopreservation versus automated volume-reduced CBUs (processed since 2005) with a mean of 88.64.91 ±â€…3.91% after 18 years of cryopreservation. In addition, these relevant parameters were retrospectively analyzed for released transplants in correlation to the storage time. Moreover, the follow-up data of recipients from CBUs cryopreserved directly (unseparated) versus CBUs cryopreserved after manual versus automated volume-reduction are presented here demonstrating an earlier engraftment in both volume-reduced groups as compared to unseparated CBUs. By this retrospective analysis, key questions are discussed regarding cord blood parameters in relation to processing methods, engraftment, and patient age (children and adults).

Multicenter evaluation of the IL-3-pSTAT5 assay to assess the potency of cryopreserved stem cells from cord blood units: The BEST Collaborative study.

BACKGROUND: The IL-3-pSTAT5 assay, a new, rapid, and standardized flow-cytometry-based assay may compensate for several limitations of the colony-forming unit (CFU) assay typically used for stem cell potency assessments of cord blood units (CBU). We performed an inter-laboratory evaluation of the performance of this new assay. STUDY DESIGN AND METHODS: This Biomedical Excellence for Safer Transfusion (BEST) Collaborative multicenter, international study included 15 participants from public cord blood banks (CBBs), CBB-supporting research laboratories, and stem cell laboratories. To perform the IL-3-pSTAT5 assay, participating centers received reagents, instructions, and 10 blind CBU samples, including eight normal samples and two samples exposed to a transient warming event. We measured inter-laboratory agreement qualitatively (proportion of correctly classified samples) and quantitatively (coefficient of variation [CV], correlation coefficients, receiver operating characteristics (ROC) curve, and intraclass correlation coefficient [ICC]). RESULTS: The qualitative agreement was 97.3% (i.e., 107/110; Fleiss' kappa = 0.835). The average CV on a per-sample basis was 11.57% among all samples, 8.99% among normal samples, and on a per-center basis was 9.42% among normal samples. In a correlation matrix that compared results across centers, the mean Pearson's correlation coefficient was 0.88 (standard deviation = 0.04). The ICC was 0.83 (95% confidence interval = 0.68-0.95). The area under the curve (AUC) from the ROC curve was 0.9974. DISCUSSION: Excellent qualitative and quantitative agreement was exhibited across laboratories. The IL-3-pSTAT5 assay may therefore be implemented in flow cytometry laboratories to rapidly and reliably provide standardized measures of stem cell potency in CBUs.

GMP-grade CD34+ selection from HLA-homozygous licensed cord blood units and short-term expansion under European ATMP regulations.

BACKGROUND: Based on a synergistic consortium, the cord blood (CB) bank Düsseldorf was responsible for the selection of HLA-homozygous (HLA-h) donors, contacting/re-consenting the mothers, Good Manufacturing Practice (GMP)-grade CD34+ enrichment, followed by short-term expansion of CD34+ cells and qualification of the resulting CD34+ population as advanced therapy medicinal product (ATMP)-starting material. Among 20 639 licensed Düsseldorf cord blood units (CBUs), 139 potential HLA-h donors were identified with the most frequent 10 German haplotypes. 100% of the donors were contacted, and for 47·5%, consent was obtained. HLA-A, -B, -C, -DR, -DQ and -DP were determined by sequencing. METHODS: Thawing/washing of the CBUs was performed in the presence of Volulyte/HSA with Sepax® , CD34+ selection by automated CliniMACS® -system (Miltenyi), expansion with qualified GMP-grade cytokines and media in the GMP facility. RESULTS: Here, we specify minimal criteria (≥5 x 105 viable CD34+ -count, ≥80% CD34+ -purity and ≥70% viability) and confirm that n = 10 CB units (max storage time 16 years) could be qualified for an ATMP starting material. The mean fold change expansion of isolated CD34+ cells at Day 3/4 (d3/4) was 3·38 ± 3·02 with a mean purity of 86·90 ± 10·38% and a high viability of 96·07 ± 4·72%. CONCLUSION: As of March 2019, approval was obtained by the Bezirksregierung Düsseldorf for the GMP-compliant production. The production of HLA-homozygous expanded CD34+ cells from cryopreserved CB under European ATMP regulations presented here describes the successful clinical translation and implementation of a qualified manufacturing process. This approach considers the main obstacle of rejection of transplanted cells (due to the immunological HLA barrier) by preselection of HLA-homozygous transplants.

Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units.

BACKGROUND AIMS: In 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards. METHODS: Twelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures. RESULTS: Excellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells. CONCLUSIONS: The current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.

The influence of temperature treatment before cryopreservation on the viability and potency of cryopreserved and thawed CD34+ and CD45+ cord blood cells.

BACKGROUND: Hematopoietic stem cell (HSC) viability and potency is crucial for qualified cord blood (CB) transplants. This study analyzes time and temperature condition before cryopreservation for the viability of CD34+/CD45+ cells after cryopreservation. METHODS: Cell viabilities were determined by antibody co-staining with 7-aminoactinomycin D detecting necrotic cells, and subsequent flow cytometric analysis. Additionally, Annexin V staining for determination of apoptotic cells and colony-forming unit (CFU) assays for testing functional potency of HSCs were performed. RESULTS: For all cell types assessed (CD45+/CD34+ cells, lymphocytes and granulocytes), the highest viabilities were obtained for CB maintained at 4°C or room temperature (RT; 22 ± 4°C) and cryopreserved directly after collection. Starting material were CB units with an age of 24.7 ± 3.5 h after birth. Post-thaw CD34+ cell results were > 90% after temperature treatment of t = 24 h (48 h total age) and > 70% after t = 48 h (72 h total age) at 4°C (48 h, 91.4 ± 5.5%; 72 h, 75.0 ± 12.0%) and RT (48 h, 84.2 ± 9.7%; 72 h, 72.6 ± 0.6%). Viabilities for 30°C samples were < 80% after t = 24 h (48 h total age, 79.8 ± 3.1%) and < 50% after t = 48 h of treatment (72 h total age, 46.8 ± 14.3%). Regarding CFU recovery of pre-freeze (without volume reduction) and thawed CB, a trend toward the highest recoveries was observed at 4°C/RT. The difference between 4°C (77.5 ± 12.0%) and 30°C samples (53.9 ± 4.8%) was shown to be significant in post-thaw samples after t = 24 h treatment (48 h total age; P = 0.0341). DISCUSSION: Delays between collection and cryopreservation should be minimized because increasing time reduces numbers of viable cells and CFUs before/after cryopreservation. CB units should be maintained at 4°C/RT to retain the highest possible potency of the cells after thawing.

Low oxygen tension reveals distinct HOX codes in human cord blood-derived stromal cells associated with specific endochondral ossification capacities in vitro and in vivo.

Effects of oxygen tension on the generation, expansion, proliferation and differentiation of stromal cell types is widely described in the literature. However, data on the internal heterogeneity of applied cell populations at different O2 levels and possible impacts on differentiation potentials are controversial. Here, the expression of 39 human HOX genes was determined in neonatal cord blood stromal cells and linked to differentiation-associated signatures. In cord blood, unrestricted somatic stromal cells (USSCs), lacking HOX gene expression, and cord blood-derived multipotent stromal cells (CB-MSCs), expressing about 20 HOX genes, are distinguished by their specific HOX code. Interestingly, 74% of the clones generated at 21% O2 were HOX-negative USSCs, whereas 73% of upcoming clones at 3% O2 were HOX-positive CB-MSCs. In order to better categorize distinct cell lines generated at 3% O2 , the expression of all 39 HOX genes within HOX clusters A, B, C and D were tested and new subtypes defined: cells negative in all four HOX clusters (USSCs); cells positive in all four clusters (CB-MSCsABCD ); and subpopulations missing a single cluster (CB-MSCsACD and CB-MSCsBCD ). Comprehensive qPCR analyses of established chondro-osteomarkers revealed subtype-specific signatures verifiably associated with in vitro and in vivo differentiation capacity. The data presented here underline the necessity of better characterizing distinct cell populations at a clonal level, taking advantage of the inherent specific HOX code as a distinguishing feature between individual subtypes. Moreover, the correlation of subtype-specific molecular signatures with in vitro and in vivo bone formation is discussed. Copyright © 2016 John Wiley & Sons, Ltd.

Prevention report of Germany's National Prevention Conference - Goals and possibilities of the initial 2019 report.

The Preventive Health Care Act Germany adopted in July 2015 defines that the institutions involved in the National Prevention Conference have to publish a prevention report on a four year basis. In the report they have to detail their efforts towards settings-based primary prevention and health promotion. This article outlines the legal requirements for the prevention report and the status of the concept for the first report in 2019.

No Identical "Mesenchymal Stem Cells" at Different Times and Sites: Human Committed Progenitors of Distinct Origin and Differentiation Potential Are Incorporated as Adventitial Cells in Microvessels.

A widely shared view reads that mesenchymal stem/stromal cells ("MSCs") are ubiquitous in human connective tissues, can be defined by a common in vitro phenotype, share a skeletogenic potential as assessed by in vitro differentiation assays, and coincide with ubiquitous pericytes. Using stringent in vivo differentiation assays and transcriptome analysis, we show that human cell populations from different anatomical sources, regarded as "MSCs" based on these criteria and assumptions, actually differ widely in their transcriptomic signature and in vivo differentiation potential. In contrast, they share the capacity to guide the assembly of functional microvessels in vivo, regardless of their anatomical source, or in situ identity as perivascular or circulating cells. This analysis reveals that muscle pericytes, which are not spontaneously osteochondrogenic as previously claimed, may indeed coincide with an ectopic perivascular subset of committed myogenic cells similar to satellite cells. Cord blood-derived stromal cells, on the other hand, display the unique capacity to form cartilage in vivo spontaneously, in addition to an assayable osteogenic capacity. These data suggest the need to revise current misconceptions on the origin and function of so-called "MSCs," with important applicative implications. The data also support the view that rather than a uniform class of "MSCs," different mesoderm derivatives include distinct classes of tissue-specific committed progenitors, possibly of different developmental origin.

The effects of culture conditions on the functionality of efficiently obtained mesenchymal stromal cells from human cord blood.

BACKGROUND AIMS: Cord blood (CB) is an attractive source of mesenchymal stromal cells (MSCs) because of its abundant availability and ease of collection. However, the success rate of generating CB-MSCs is low. In this study, our aim was to demonstrate the efficiency of our previously described method to obtain MSCs from CB and further characterize them and to study the effects of different culture conditions on MSCs. METHODS: CB-MSC cultures were established in low oxygen (3%) conditions on fibronectin in 10% fetal bovine serum containing culture medium supplemented with combinations of growth factors. Cells were characterized for their adipogenic, osteogenic and chondrogenic differentiation capacity; phenotype; and HOX gene expression profile. The functionality of the cells cultured in different media was tested in vitro with angiogenesis and T-cell proliferation assays. RESULTS: We demonstrate 87% efficacy in generating MSCs from CB. The established cells had typical MSC characteristics with reduced adipogenic differentiation potential and a unique HOX gene fingerprint. Growth factor-rich medium and a 3% oxygen condition enhanced cell proliferation; however, the growth factor-rich medium had a negative effect on the expression of CD90. Dexamethasone-containing medium improved the capacity of the cells to suppress T-cell proliferation, whereas the cells grown without dexamethasone were more able to support angiogenesis. CONCLUSIONS: Our results demonstrate that the composition of expansion medium is critical for the functionality of MSCs and should always be appropriately defined for each purpose.

RNA Amplification Protocol Leads to Biased Polymerase Chain Reaction Results Especially for Low-Copy Transcripts of Human Bone Marrow-Derived Stromal Cells.

The amplification of RNA is becoming increasingly important, as often only limited amounts of cells are available for gene expression analysis. In this study, the gene expression profile of the 39 human homeobox (HOX) genes was analyzed in bone marrow-derived multipotent stromal cells (BM-MSCs) by reverse transcription (RT-) and quantitative polymerase chain reaction (qPCR). For further unlimited gene expression analysis, Whole Transcriptome Amplification (WTA) was used to amplify RNA from human BM-MSCs. However, WTA led to biased RT- and qPCR results, and even non-detectability of HOX transcripts compared to non-amplified BM-MSC samples which instead revealed transcription. It is important to note that the same RNA of the respective human BM-MSC line was used for normal cDNA synthesis by standard reverse transcription (non-amplified RT samples) and for cDNA synthesis by WTA (amplified WTA samples). On this account, the different RT- and qPCR results were unexpected applying WTA. The significantly reduced detection of HOX transcripts after WTA has been demonstrated for numerous BM-MSC lines (n = 26) by RT-PCR analysis. Furthermore, undetectable HOX transcripts meaning HOX transcripts of human BM-MSCs that were detected after normal cDNA synthesis, but were no longer detectable after WTA, were consistently observed by qPCR analysis. Finally, qPCR experiments revealed a possible explanation for the differences between amplified and non-amplified BM-MSC samples: an inverse correlation between the biased qPCR results and the low expression level of the respective HOX gene. The PCR analysis of high-copy transcripts like GAPDH or RPL13A revealed unchanged qPCR results after WTA compared to corresponding non-amplified BM-MSC samples. In contrast, WTA led to biased qPCR results for medium-copy HOX transcripts, and even non-detectability of low-copy HOX transcripts of human BM-MSCs resulting in false negative RT- and qPCR data applying WTA.

DNA damage response in neonatal and adult stromal cells compared with induced pluripotent stem cells.

UNLABELLED: Comprehensive analyses comparing individual DNA damage response (DDR) of induced pluripotent stem cells (iPSCs) with neonatal stromal cells with respect to their developmental age are limited. The imperative necessity of providing developmental age-matched cell sources for meaningful toxicological drug safety assessments in replacement of animal-based testing strategies is evident. Here, DDR after radiation or treatment with N-methyl-N-nitrosurea (MNU) was determined in iPSCs compared with neonatal and bone marrow stromal cells. Neonatal and adult stromal cells showed no significant morphologically detectable cytotoxicity following treatment with 1 Gy or 1 mM MNU, whereas iPSCs revealed a much higher sensitivity. Foci analyses revealed an effective DNA repair in stromal cell types and iPSCs, as reflected by a rapid formation and disappearance of phosphorylated ATM and γH2AX foci. Furthermore, quantitative polymerase chain reaction analyses revealed the highest basic expression level of DDR and repair-associated genes in iPSCs, followed by neonatal stromal cells and adult stromal cells with the lowest expression levels. In addition, the influence of genotoxic stress prior to and during osteogenic differentiation of neonatal and adult stromal cells was analyzed applying common differentiation procedures. Experiments presented here suggest a developmental age-dependent basic expression level of genes involved in the processing of DNA damage. In addition a differentiation-dependent downregulation of repair genes was observed during osteogenesis. These results strongly support the requirement to provide adequate cell sources for toxicological in vitro drug testing strategies that match to the developmental age and differentiation status of the presumptive target cell of interest. SIGNIFICANCE: The results obtained in this study advance the understanding of DNA damage processing in human neonatal stromal cells as compared with adult stromal cells and induced pluripotent stem cells (iPSCs). The data suggest developmental age-dependent differences in DNA damage repair capacity. In iPSCs (closest to embryonic stem cells), the highest expression level of DNA damage response and repair genes was found, followed by neonatal stromal cells and adult stromal cells with the lowest overall expression. In addition, a differentiation-dependent downregulation of repair capacity was observed during osteogenic differentiation in neonatal stromal cells. Notably, the impact of genotoxic stress on osteogenic differentiation depended on the time the genotoxic insult took place and, moreover, was agent-specific. These results strongly support the necessity of offering and establishing adequate cell sources for informative toxicological testing matching to the developmental age and differentiation status of the respective cell of interest.

Comparing the gene expression profile of stromal cells from human cord blood and bone marrow: lack of the typical "bone" signature in cord blood cells.

With regard to the bone-regenerative capacity, bone marrow stromal cells (BMSC) can still be termed the "gold standard." Nevertheless, neonatal stromal cells from cord blood (CB) feature advantages concerning availability, immaturity, and proliferation potential. The detailed gene expression analysis and overexpression of genes expressed differentially provide insight into the inherent capacity of stromal cells. Microarray and qRT-PCR analyses revealed closely related gene expression patterns of two stromal cell populations derived from CB. In contrast to the CB-derived cell types, BMSC displayed high expression levels of BSP, OSX, BMP4, OC, and PITX2. Lentiviral overexpression of BSP but not of OSX in CB-cells increased the capacity to form a mineralized matrix. BMP4 induced the secretion of proteoglycans during chondrogenic pellet culture and extended the osteogenic but reduced the adipogenic differentiation potential. BMSC revealed the typical osteogenic gene expression signature. In contrast, the CB-derived cell types exhibited a more immature gene expression profile and no predisposition towards skeletal development. The absence of BSP and BMP4-which were defined as potential key players affecting the differentiation potential-in neonatal stromal cells should be taken into consideration when choosing a cell source for tissue regeneration approaches.

Neonatal mesenchymal-like cells adapt to surrounding cells.

Hematopoietic cord blood (CB) transplantations are performed to treat patients with life-threatening diseases. Besides endothelial cells, the neonatal multipotent stromal cell subpopulations CDSCs (CB-derived stromal cells) and USSCs (unrestricted somatic stromal cells) are like bone marrow (BM) SCs interesting candidates for clinical applications if detailed knowledge is available. Clonal USSC compared to CDSC and BMSC lines differ in their developmental origin reflected by a distinct HOX expression. About 20 (out of 39) HOX genes are expressed in CDSCs (HOX+), whereas native USSCs reveal no HOX gene expression (HOX-). Moreover, USSCs display a lineage-specific absence of the adipogenic differentiation potential. As the specific HOX code can be ascribed to topographic bodysites it may be important to match the HOX code of transplanted cells to the tissue of interest. Herein co-culture experiments were performed, presenting a novel approach to modulate the differentiation potency of USSCs towards HOX positive stromal cells. After co-culturing native USSCs with CDSCs and BMSCs, USSCs adapt a positive HOX code and gain the adipogenic differentiation capacity. These results present for the first time modulation of a lineage-specific differentiation potential by co-culture. Finally, USSCs can be claimed as potential candidates to substitute unique progenitor cell populations in clinical approaches.

Portal application of human unrestricted somatic stem cells to support hepatic regeneration after portal embolization and tumor surgery.

Insufficient liver remnant volume still precludes patients with potentially resectable tumors from curative surgery. Clinically, it has been demonstrated that transplanted adult stem cells promote liver regeneration. However, the mechanisms of the observed functional improvements are unknown. The aim of our study was to evaluate the impact of transplanted human multipotent cord blood-derived unrestricted somatic stem cells (USSC) on liver regeneration and identify the underlying mechanisms in an ovine model. We performed partial embolization of the right liver lobe and grafted USSC in the portal venous system of the left liver lobe. After 4 weeks, livers were explanted and analyzed for differentiation of USSC into hepatocytes by histopathologic examination and for fusion of USSC with recipient hepatocytes by single-cell polymerase chain reaction. The studies revealed that transplanted USSC differentiate into hepatocytes and produce human albumin. No ovine DNA was found in the hepatocytes with a human phenotype. Transplantation of USSC enhances the number of viable hepatocytes in liver disease by differentiation and opens new therapeutic perspectives.

Oxygen tension modifies the 'stemness' of human cord blood-derived stem cells.

BACKGROUND AIMS: Amongst different stem cell populations derived from human cord blood (CB), unrestricted somatic stem cells (USSC) are distinguished from CB mesenchymal stromal cells (CB MSC) by expression patterns of homeobox (HOX) genes, delta-like1 homolog (DLK1) expression and adipogenic differentiation potential. In this study we investigated the effects of oxygen tension on the generation, proliferation and expression of stem cell marker genes, which could be critical during large-scale cell culture for clinical applications. METHODS: We cultured CB-derived stem cells at 5% and 20% O(2). Telomere length shortening was analyzed and we investigated gene expression using reverse-transcription (RT)-polymerase chain reaction (PCR) and real-time PCR. Additionally we performed adipogenic and osteogenic in vitro differentiation. Results. Altering the cultivation conditions of USSC or CB MSC from 20% to 5% O(2) had no significant impact. In contrast, cell populations derived from primary cultures prepared at 5% O(2) qualified as neither USSC nor as CB MSC. When converted to 20%, their proliferation was diminished, telomere shortening was accelerated, and two of six cell lines ceased expression of HOX genes. The HOX code of the other cell populations was not been affected by culture conditions. CONCLUSIONS: Altering culture conditions during generation can impact cell characteristics such as the HOX code. These effects need to be considered when dealing with cell cultures for clinical applications.

Postmenopausal sex hormones in relation to body fat distribution.

Being overweight or obese increases the risk of postmenopausal breast cancer. A potential reason may be the frequently observed positive association of BMI with endogenous sex hormones and its negative association with sex hormone-binding globulin (SHBG). The purpose of this study was to investigate whether a woman's body fat distribution shows a BMI-independent association with these breast cancer-related biomarkers. Performing cross-sectional analyses among 1,180 postmenopausal women, we assessed whether associations of surrogates for an abdominal (waist circumference; waist-to-hip ratio, WHR) and gluteofemoral (hip circumference) fat distribution with estrone, total and free estradiol, androstenedione, total and free testosterone, and SHBG changed after adjustment for, or stratification by, BMI. All anthropometric measures were positively associated with estrogens and free testosterone, and negatively with SHBG. After adjustment for BMI, associations of free estradiol, free testosterone, and SHBG with both waist circumference and WHR remained significant, but all initially significant associations with hip circumference were abolished. In stratified analyses, waist circumference and WHR correlated with free estradiol, free testosterone, and SHBG in women with a BMI < 30 kg/m(2) but not in women with a BMI ≥ 30 kg/m(2). The latter suggests that in obese women, a possibly unique effect of abdominal fat on these biomarkers may be masked by the already large amount of overall body fat. On the whole, our results indicate that waist circumference and WHR, but not hip circumference, are associated with SHBG and SHBG-related sex hormones (free estradiol and free testosterone) independently of BMI.

Distinct differentiation potential of "MSC" derived from cord blood and umbilical cord: are cord-derived cells true mesenchymal stromal cells?

Mesenchymal stromal cells (MSC) with distinct differentiation properties have been reported in many adult [eg, bone marrow (BM)] or fetal tissues [eg, cord blood (CB); umbilical cord (UC)] and are defined by their specific surface antigen expression and multipotent differentiation potential. The MSC identity of these cells should be validated by applying well-defined readout systems if a clinical application is considered. In order to determine whether cells isolated from human UC fulfill the criteria defined for MSC, the immunophenotype and differentiation potential including gene expression analysis of the most relevant lineage-specific markers were analyzed in the presented report in combination with the HOX-gene expression. Cells from the UC do not differentiate into osteoblasts demonstrated by Alizarin Red and Von Kossa staining in addition to real-time polymerase chain reaction (PCR)-analysis of runt-related transcription factor 2, bone sialoprotein, osteocalcin, osterix, bone morphogenetic proteins 2 and 4. Oil Red O staining as well as PCR analysis of peroxisome proliferator-activated receptor-gamma, fatty acid-binding protein 4, and perilipin revealed an absent adipogenic differentiation. The lack of potential to differentiate into chondrocytes was documented by Alcian-Blue periodic acid-Schiff, Safranin O staining, and real-time PCR analysis of SOX9. Furthermore, neither endothelial nor myogenic differentiation was documented after induction of UC-MSC. In comparison to CB- and BM-derived cells, UC cells revealed an absent trilineage differentiation capacity in vitro. Therefore, these cells should not be termed "mesenchymal stromal cells". The UC cells can be distinguished from CB- and BM-derived cells as well as from pericytes and foreskin fibroblasts by the expression of HOX-genes and the cell surface antigens CD56 and CD146.

Physical activity and endogenous sex hormones in postmenopausal women: to what extent are observed associations confounded or modified by BMI?

OBJECTIVES: To investigate associations between physical activity and endogenous sex hormones after menopause with a special focus on confounding and effect modification by body mass index (BMI). METHODS: A cross-sectional study among 1,260 postmenopausal women was conducted. Generalized linear models were used to compare levels of total leisure-time physical activity, sports activities, bicycling, and walking with levels of sex hormones and sex-hormone-binding-globulin (SHBG). RESULTS: Higher sports activity levels were significantly associated with lower levels of estrone and total and free testosterone in multivariate adjusted models. After additional adjustment for BMI, associations with estrone and free testosterone were attenuated; the association with total testosterone remained unchanged. No physical activity variable was significantly related to total and free estradiol, androstenedione, or SHBG. We did not observe effect modification by BMI. CONCLUSIONS: Sports activities may lead to lower levels of estrone and testosterone in postmenopausal women. While effects on estrone and free testosterone seem to be largely mediated by BMI, effects on total testosterone appear to be mainly independent of BMI. The BMI-independent effects on these hormones (especially on total testosterone) could at least partly explain why physical activity has been frequently reported to be preventive for postmenopausal breast cancer, even after accounting for BMI.